Electrode for the electrochemical detection of nitric oxide

ABSTRACT

Nitric oxide-specific electrodes are useful for in situ detection of nitric oxide in biomedical applications and have at least a surface region thereof which is capable of forming complexes with nitric oxide, for example, nitrosyl complexes. The nitric oxide complexes formed at the surface of the electrodes apparently increase the concentration of nitric oxide available for detection, thereby leading to significantly improved relative responses as compared to other known nitric oxide electrode materials. Most preferably, the electrode has at least an exterior surface region which contains ruthenium and/or at least one oxide of ruthenium. The electrodes are advantageously conditioned in saline solution at +675 mV for about two hours.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Application Ser. No.08/942,354 filed on Oct. 1, 1997 (now U.S. Pat. No. 5,980,705), and isbased on, and claims domestic priority benefits under 35 USC §119(e)from, U.S. Provisional Application Ser. No. 60/027,355 filed on Oct. 2,1996, the entire content of each application being expresslyincorporated hereinto by reference.

GOVERNMENT GRANT STATEMENT

This invention was made with Government support under Grant No. 2 PO1HL42444-06 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention generally relates to the field of electrochemicaldetection of nitric oxide. In preferred embodiments, the presentinvention relates to electrodes and microelectrodes having improvedresponses to nitric oxide by chemically increasing the concentration ofnitric oxide available for detection at the electrode surface and/orcatalyzing the electrolysis of nitric oxide.

BACKGROUND OF THE INVENTION

Nitric oxide has just recently been identified as a molecule which playsa fundamental role in biological processes. As a result, research intothe physiology and pathology of nitric oxide has grown explosively. Thisresearch activity has, in turn, created a demand for accurate andprecise techniques for the determination of nitric oxide (NO.), a freeradical gas that is short-lived in biological materials.

Several methods for detecting nitric oxide in biology and medicine arewell established. These include spectrophotometry, chemiluminescence,and paramagnetic resonance. These are ex situ techniques, however. Thatis, a sample of biological fluid, for example the extracellular fluid ina tissue or the support buffer in a suspension of cells, must beanalyzed out of its biological context. The measurements made on suchsamples reflect nitric oxide concentration at a single time, and whenassembled in a series make a discontinuous record. Therefore, thesemethods, though valuable, are not ideal for following rapid processes,because changes in nitric oxide concentration are not observed if theyoccur between sampling points. However, the ability to follow rapidchanges is important because nitric oxide is unstable in the presence ofoxygen, persisting but a few minutes or seconds in biological systems.

Recently, electrodes for the direct electrochemical detection of nitricoxide have been developed. The earliest of these electrodes, knowncolloquially as the “Shibuki electrode”, is a modification of anelectrode for detecting O₂ and functions to exclude interfering speciesby means of a membrane permeable only to gases. See, K. Shibuki,Neuroscience Research, 9 (1990) 69-76 (the entire content of which isexpressly incorporated hereinto by reference). The Shibuki electrodeuses a Pt electrode to oxidize nitric oxide at 800 mV and to registerthe resulting oxidation current. This sensor is reported to have limitedbiological usefulness, because it does not respond linearly to nitricoxide concentrations greater than 1 μM and is subject to a destructivebuildup of the oxidation products of nitric oxide within the enclosedelectrolyte surrounding the Pt electrode.

More recently, another method became available, using a metalloporphyrinmembrane electrochemically deposited on a carbon fiber electrode. See,T. Malinski et al, Nature, vol. 358, 676-677 (1992); T. Malinski et al,“Nitric Oxide Measurement by Electrochemical Methods”, Methods in NitricOxide Research, chapter 22 (1996); and Published International PatentApplication No. WO 93/21518 to T. Malinski (the entire content of eachpublication being expressly incorporated hereinto by reference). Thissensor is constructed by electrochemically depositing ametalloporphyrin, for example nickel-tetrakis (3-methoxy-4hydroxyphenyl)porphyrin, on a carbon electrode (which may be as small as a singlecarbon fiber a few μm in diameter, or less). The porphyrin surface isthen coated with a final layer of Nafion™ (Dupont), a fluorocarbonpolymer that forms a network of interconnected cavities lined withsulfonate groups (SO₃ ⁻). Cations and neutral solutes are conductedthrough the cavities with the anions being excluded. Direct measurementsof nitric oxide have been reported using this porphyrinized electrode,with good sensitivity and selectivity. Furthermore, this porphyrinizedelectrode can be produced in micron or submicron tip diameters, suitablefor extra- and intra-cellular measurements. Its disadvantages includethe difficulty of handling micron-diameter carbon fibers, which requiremanipulation under a microscope with cold illumination (or under water)to eliminate thermal convection currents that disturb the fibers.Furthermore, the fibers, though strong for their size, are easily brokenand are not degraded or absorbed in biological tissue. There areadditional concerns about the exact chemical mechanism by which thiselectrode detects nitric oxide, since carbon fibers without a porphyrincoating or with a coating of porphyrin without a metal ligand can alsodetect nitric oxide with significant sensitivity.

A biochemically-modified electrode has also been proposed that employsCytochrome c as a nitric oxide sensor, catalyzing the electrochemicalreduction of nitric oxide and NO₂ ⁻ at −580 mV. See, K. Miki et al,Journal of Electroanalytical Chemistry, 6, 703-705 (1993) (the entirecontent of which is expressly incorporated hereinto by reference).

Nitric oxide-detecting electrodes have also been constructed with wiresof precious metals, notably platinum and gold. See, F. Pariente et al,“Chemically modified electrode for the selective and sensitivedetermination of nitric oxide (NO) in vitro and in biological systems”,Journal of Electroanalytical Chemistry, 379, 191-197 (1994) and F.Bedioui et al, “The use of gold electrodes in the electrochemicaldetection of nitric oxide in aqueous solution”, Journal ofElectroanalytical Chemistry, 377, 295-298 (1994) (the entire content ofeach publication being hereby expressly incorporated hereinto byreference). Furthermore, a porphyrinic-based platinum-iridium electrodehas also been constructed and is available commercially. See, K.Ichimori et al, “Practical nitric oxide measurement employing a nitricoxide-selective electrode”, Ref. Sci. Instrum., 65 (8) August 1994 andH. Miyoshi, FEBS Letters, 345, 47-49 (1994) (the entire content of eachpublication being hereby expressly incorporated hereinto by reference).

Thus, although there have been prior proposals in the literature, thedevelopment of electrodes for the electrochemical detection of nitricoxide has yet to reach the level that would allow widespread use inbiomedical research. Such an electrode must be: (1) highly sensitive(with limits of detection for nitric oxide in the nanomolar range andbelow); (2) highly selective for nitric oxide against interferinganions; (3) easy to prepare reproducibly in very small diameters (10 μmor less); (4) able to respond rapidly to nitric oxide, whose half-lifeis but a few seconds in physiological conditions; and (5) stable forminutes to hours in biological fluids and tissues. It is towardsfulfilling such needs that the present invention is directed.

SUMMARY OF THE INVENTION

Broadly, the present invention relates to electrodes which exhibit anincrease in electrical current due to the electrolysis of nitric oxideeither by increasing the concentration of nitric oxide available at theelectrode surface or by increasing the rate constant of the electrolysisat a given potential. More specifically, the electrodes of thisinvention have a surface region prepared from a material (for example,the element ruthenium) which is known to be capable of forming complexeswith nitric oxide when exposed to nitric oxide. Such complexes mayinclude nitrosyl complexes, M—NO (where M is a metal), isonitrosylcomplexes, M—ON, nitrosyl-isonitrosyl complexes, M—NO—M′ (where M′ ismetal that may or may not be the same as M), and NO complexes involvingboth metals and non-metals (including, but not limited tometal-nitrosyl-chloro complexes, such as ruthenium-nitrosyl-chlorocomplexes), and any other complexes involving one or more molecules ofNO (in any of its oxidation states) with one or more other atoms ormolecules. It is presently believed that the formation of such nitrosylcomplexes will increase the selectivity of an electrode to nitric oxideover other substances because of the specificity of the process by whichnitrosyl complexes are formed. In addition, the present invention offersthe possibility of indirect detection of nitric oxide, since undercertain conditions the formation of complexes with nitric oxide on theelectrode surface may inhibit the electrolysis of other electroactivespecies (e.g., chloride, Cl⁻), thereby causing a detectable change inbackground current. This change in background current may besignificantly larger than the current due to direct oxidation orreduction of nitric oxide.

Most preferably, the electrodes of this invention include ruthenium,which in and of itself is an element known to form the greatest numberof distinct nitrosyl complexes. (See, Griffith, The Chemistry of TheRarer Platinum Metals, Interscience Publishers, pp. 174-175 (1967), theentire content of which is expressly incorporated hereinto byreference.) The electrodes of this invention may thus be prepared fromruthenium, or have a coating prepared from ruthenium on a core ofsupporting material. Alternatively, the ruthenium may be combined withone or more metals or non-metals as may be desired.

Included within the scope of this invention are other materials that maybe devised which also enhance the electrolysis of nitric oxide by meansof a significant ability to form complexes with nitric oxide. Suchmaterials include metals, their inorganic compounds, and organiccompounds, including but not limited to elements and compounds describedin G.B. Richter-Addo et al, Metal Nitrosyls, Oxford University Press(1992), the entire content of which is expressly incorporated hereintoby reference. Examples include, but are not limited to, Ti, Co, W, andother transition metals.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Reference will hereinafter be made to the accompanying drawing FIGURES,wherein:

FIG. 1 is a bar graph plot showing the relative responses to nitricoxide of 0.5 mm diameter disk electrodes made of various materials,including an electrode according to the present invention, wherein theresponse of a carbon electrode is arbitrarily equal to 1, and whereinC=carbon, C/por=carbon coated with a nickel porphyrin, pt=platinum,Pd=palladium, Ir=iridium, Rh=rhodium and Ru=ruthenium;

FIG. 2 is a graphical plot of the difference in current between twolinear sweep voltamograms using a 0.5 mm ruthenium electrode in thepresence and the absence, respectively, of nitric oxide;

FIG. 3 is a graphical plot which shows the results of the cyclicvoltammetric results of the second series of experiments described belowin Example II; and

FIG. 4 is a graphical plot showing the detection of 1.5 μM nitric oxideadded to human blood plasma.

DETAILED DESCRIPTION OF THE INVENTION

The electrodes of this invention can be incorporated into a broadvariety of biomedical and/or clinical medical devices to facilitatebiomedical research and to enable the accurate and/or early diagnosisand monitoring of various medical conditions and/or disease states inwhich nitric oxide plays a role. For example, the electrodes of thisinvention may be incorporated into catheters, needles, cannulas and thelike for insertion into a patient or experimental subject (e.g.,intravenously, into a synovial capsule (joint capsule), into thecerebrospinal fluid, or through a natural body orifice, such as theurethra). The electrode may thus be brought into proximity to aparticular biological fluid, such as blood, urine, synovial fluid andthe like to allow for the real time detection in vivo of nitric oxidetherein. The electrodes of this invention may therefore be employedusefully for the diagnosis and/or monitoring of bacterial infections(e.g., sepsis), autoimmune diseases (e.g., rheumatoid arthritis) and anyother disease or condition in which nitric oxide plays a role. Sufficeit to say here that the electrodes of this invention may also beincorporated into patient monitoring equipment (e.g., blood drawingsystems) or laboratory bench devices.

A greater understanding of this invention will be gained by thefollowing Examples.

EXAMPLE I

Reagents: Phosphate buffered saline (PBS) was prepared by dissolvingNaCl (to a final concentration of 150 mM) in 7.0 pH phosphate buffer(Fisher). Nafion™ perfluorinated ion-exchanger resin (5 wt % in loweraliphatic alcohols and water) was purchased from Aldrich ChemicalCompany and used as supplied. N₂ (Ultra High Purity Grade, containingless than 5 micromolar O₂) was supplied by National Welders; and 10% NO.in N₂ (actual analysis: 9.98% NO., balance N₂) was supplied by NationalSpecialty Gases. Nickel(II)-Tetrakis(3-methoxy-4hydroxyphenyl) porphyrin(Ni-TMHPP) was supplied by Midcentury Chemical Co. (Posen, Ill.) andused without further purification.

Instrumentation and Electrodes: The electrochemical responses of testelectrodes were observed with a model BAS 100-B potentiostat equippedwith a preamplifier, model PA-1, and an electrically-shielded cellstand, model CS-2, all manufactured by Bioanalytical Systems (WestLafayette, Ind.). Data were recorded on a personal computer.

Disk electrodes were fabricated in the laboratory from 1-cm lengths ofmetal wire or carbon rod. The following precious metals and alloys weresupplied by Aldrich Chemical Company: Ir (99.9%), Pd (99.99%), Pt(99.99%), and Rh (99.9%). Centerless ground ruthenium slugs (99.9%) weresupplied by Englehard Corp. Carbon rod was improvised from mechanicalpencil refills consisting of carbon particles in a polymer binder, asmanufactured by Scripto (Ultrapolymer Leads™, HB hardness grade). Allelectrode materials were 0.5 mm in diameter and in the form of a roundsolid cylinder, except for the 99.9% Iridium, which is difficult to drawas a round wire (Mohs hardness 6.5). Thus, Iridium was supplied as asolid square cylinder, 0.5 mm on a side.

Electrical leads were prepared by stripping 1 cm of electricalinsulation from each end of an 8 cm length of 30 AWG solid copper wireinsulated with Kynar™, which withstands temperatures above 300° C.(Radio Shack). Each electrode slug was wrapped tightly with strippedcopper wire. The electrode and its electrical lead were then insertedinto a 6 cm length of shrink-melt tubing, a tetrafluoroethylene (TFE)tube with a fluorinated ethylene propylene (FEP) lining (Small PartsCo.), with the tip of electrode flush with one end of the tubing and theexcess lead wire protruding from the other end. This assembly was heatedto approximately 350° C. with a thermostatically-controlled heat gununtil the TFE tubing shrunk snugly around the electrode and the FEPlining melted to transparency.

After encapsulation, a disk of the electrode material was exposed bycutting through the melted tubing as close to the metal electrode tip aspossible. This exposed disk was polished, first on 600 grit siliconcarbide abrasive paper (Buehler) in water, and then successively withaqueous slurries of powered alumina abrasive (Buehler) of 0.5, 0.3, 0.1and 0.05 μm particle size. Electrodes were cleaned ultrasonically inde-ionized water after each polishing step. After polishing wascompleted, each electrode was given a final ultrasonic cleaning inmethanol. The integrity of the seal of the FEP liner around theelectrode was later tested by observing repeated cyclic voltamograms inpotassium ferricyanide solution. The near superposition of repeatedvoltamograms after 10 cycles was taken to indicate that analyte did notcreep by capillarity between the electrode material and itsencapsulation, thereby increasing the effective surface area of theelectrode. The electrical resistance of the electrode assembly measuredless than 0.5 Ω in all cases, even after immersion in buffered salinesolution for two weeks, suggesting that significant corrosion of theencapsulated connection between the copper lead wire and the electrodeslug did not occur.

Coating Carbon Electrodes with Porphyrin: Two kinds of carbon electrodeswere evaluated for purposes of comparison with the electrodes of thisinvention—these being, a carbon electrode without a porphyrin coatingand a carbon electrode with a metalloporphyrin coating. Theporphyrin-coated carbon electrode was prepared by immersing a polishedand rinsed carbon electrode in 0.5 mMNickel(II)-Tetrakis(3-methoxy-4-hydroxyphenyl) porphyrin (Ni-TMHPP) in0.1M NaOH. Cyclic Voltammetry was performed (−200 to 1200 mV at 100mV/s) at room temperature (approximately 21 ° C.) for 50 cycles. Thedeposition of successive layers of the metalloporphyrin could befollowed by observing the growth in the Ni(II)/Ni(III) oxidation peaks.

Measuring Effective Areas of Disk Electrodes: After each electrode waspolished and rinsed, it was suspended in 10 mM K₃Fe(CN)₆ in 1M KCl,along with an Ag—AgCl reference electrode (filled with 3 M KCl) and witha Pt auxiliary electrode. The solution and electrodes were contained ina glass, water-jacketed electrochemical cell maintained at 35°±1° C.Linear Scan Voltammetry was performed (from 400 to 0 mV at 1mV/s). Theplateau currents of the resulting sigmoidal current-vs-voltage curveswere calculated using an algorithm provided in the BAS software. Theeffective surface area of each disk electrode was calculated by usingthe expression:

i=4n FrDC

where:

i=Steady state current (wave current)

n=Electrons per molecule oxidized or reduced (n=1 for Fe(CN)₆ ^(3−/4−))

F=Faraday's constant

r=Radius of electrode (cm); Surface Area=πr²

D=Diffusion coefficient (for ferricyanide at 35° C., cm²/sec)

C=Concentration of ferricyanide (mol/cm³)

Coating Metal and Carbon Electrodes with Nafion™ Resin: Immediatelyafter the effective surface area of each disk electrode was determinedas described above, the electrode was dipped and agitated three times indeionized water to rinse off the ferricyanide and KCl solution and thendried in an oven at 80° C. for 10 min. The electrode was then dipped inNafion™ resin for 10 seconds after which it was again oven dried at 80°C. for 10 min. The initial Nafion™ resin dip followed by drying wasrepeated 4 times, resulting in a total of 5 coats of Nafion™ resin oneach electrode. Completed electrodes were stored overnight in room airin a loosely-covered container. Electrode tips were soaked in PBS for 24hours prior to use in order to assure complete hydration of thehygroscopic Nafion™ resin membrane.

Preparation of NO. Standard Solutions: Aqueous NO. standards wereprepared by saturating PBS with 10% NO. under anaerobic conditions. Aglass vial containing 15 mL PBS was sealed with a rubber septumpenetrated by small-diameter stainless steel HPLC tubing which extendedto near the bottom of the PBS. The septum was also penetrated by a 30gauge syringe needle to vent the headspace. The PBS was bubbled withhumidified N₂ for 20 min to lower dissolved oxygen concentration to 5ppm or less, after which it was bubbled with 10% NO. in N₂ for 20minutes before use; bubbling with 10% NO. was continued throughout eachexperiment.

Determining Electrode Response to NO.: Each test electrode was immersedin 2.5 mL PBS along with a reference and auxiliary electrode in awater-jacked electrochemical cell maintained at 35°±1° C. as alreadydescribed; however, the reference electrode was also filled with PBS(instead of 3 M KCl) in order to minimize junction potentials. Theanalyte buffer was de-aerated by bubbling for 20 minutes with N₂ thatwas humidified by passage through de-ionized water. After the initialde-aeration by bubbling, N₂ was kept flowing into the headspace abovethe buffer.

For an initial two-hour conditioning period, each test electrode washeld at a fixed potential of 800 mV with respect to the reference. Thispotential was chosen because the amperometric response to NO. increasedwith potential for the materials tested, and when various potentialsbelow 800 mV were tested, there was a decreased response to NO. On theother hand, above 800 mV there was a sharp increase in backgroundcurrent for all electrode materials, presumably due to chlorideoxidation or the electrolysis of water (evolving OH⁻). Therefore,because the electrodes invariably gave the best response at higherpotentials, and because 800 mV was as high as one could go withoutinterference from chloride or hydroxyl ions, 800 mV was selected to bethe standard potential at which all electrode materials were tested.

All this was done prior to obtaining ruthenium metal for testing, andthe behavior of ruthenium was not as expected. Though it gave a poorerresponse to NO. than other materials at 800 mV, it exhibited anexcellent response at a significantly lower potential, contrary to allother materials tested. By later testing ruthenium at a variety ofpotentials between 0 and 1,000 mV, it was discovered that the oxidationcurrent in the presence of nitric oxide reached a maximum at 675 mV intime-base amperograms. At that potential, ruthenium's response to NO.was better than that of all other materials tested at 675 mV, and evenwhen the other materials were tested at a higher potential, including800 mV, ruthenium's response at 675 mV was significantly better. It isimportant to note that for amperometry in complex, biological systems,less extreme potentials (those closer to 0) are to be preferred, becausein such systems there is always a mixture of electroactive chemicalspecies, and at higher potentials more of these species may beelectrolyzed, producing a current that could be confounded with that dueto oxidation of NO.

As described above, all electrodes were maintained at the fixedpotential (800 mV, 675 MV, or other chosen potential) for two hoursbefore the exposure to NO., in order to allow the electrode current todecay to a stable baseline. This decay is presumably due partly tocharging of the electrode-solution double layer but mostly to the slowformation of an oxide layer.

After the conditioning period, the 800 mV potential for the comparativeelectrodes, 675 mV for the invention electrode, was maintained withoutinterruption for an additional 4,000 sec (1 hr, 6 min and 40 sec, amaximum imposed by the BAS potentiostat software) while amperommetry wasperformed as various volumes of the NO. calibration solution wereinjected into the PBS solution with a Hamilton gas tight syringe (25 μL,1700 series). Injections were made at 10-minute intervals, for example,25, 20, 15, 10, 5, 25, μL.

Results: The bar graph in the accompanying FIG. 1 shows the results (andstandard deviations) obtained when testing the response of variouselectrode materials to nitric oxide (approximately 0.5 to 1.5 μM). Eachbar represents three or more experimental points. Response is currentper unit area of electrode surface per unit concentration NO., expressedrelative to the response of a carbon electrode (C). As noted above, allelectrodes were held at +800 mV vs Ag/AgCl except the rutheniumelectrode, which was held at +675 mV.

As is evident from the bar graph of the accompanying FIG. 1, theelectrode according to this invention formed of ruthenium showedconsiderable improvement over the amperometric response of otherelectrode materials. It should also be apparent that the ratio ofvariation in experimental results (standard deviation) to signalstrength is best for ruthenium.

Accompanying FIG. 2 exhibits an NO.-induced change in background currentthat is significantly larger than the current due to direct oxidation orreduction of nitric oxide. Thus, the electrodes of this invention offerthe possibility of indirect detection of nitric oxide, since undercertain conditions the formation of nitroxyl complexes on the electrodesurface may displace other electroactive species (e.g., chloride, Cl⁻),thereby causing a detectable change in background current.

EXAMPLE II

In order to confirm the conclusion that NO. forms a complex with theRu-based electrode, two series of experiments were undertaken. In thefirst series changes were probed in the surface of an electrode preparedfrom Ru that occur after exposure to a fixed potential and subsequentexposure to NO. These experiments clearly show a change in thecapacitance of the electrode after polarizing it at +675 mV for 2 hours.This change is largely reversible on introduction of NO. into thesystem. In the second series of experiments, the extent to which theoxide film on the Ru electrode can be electrochemically reduced is shownto be severely diminished when surface sites are blocked by adsorbed NO.Taken together, the data from both series of experiments lend strongsupport to the mechanism herein proposed for the enhanced sensitivity ofthe Ru electrode towards oxidation of NO., i.e. surface complexation.

When a metal electrode (e.g., Ru) is immersed in an electrolytesolution, ions in solution adsorb at the electrode/solution interface tocounterbalance any excess electronic charge on the metal, forming adouble layer. Under ideal conditions, the double layer behaves like asimple capacitor, charging and discharging as the potential differencebetween the metal electrode (e.g. Ru) and the reference electrode (e.g.Ag/AgCl) is changed. The amount of charge (Q) that can be stored on acapacitor held at a potential (E) depends on the system capacitance (C)according to:

 Q=E×C

However, when a molecule (e.g., NO.) adsorbs onto the surface of themetal electrode, the capacitance of the double layer must change. Thisis analogous to changing the dielectric constant of the material betweenthe plates of a simple parallel-plate capacitor. The capacitance (C) isfirst-order with respect to the dielectric constant (e) of the mediumbetween the plates as given by:

C=(e×e ₀)/d

where (e₀) is the permittivity of vacuum, and d is the spacing betweenthe plates of the capacitor. Thus, measuring the capacitance of anelectrode in an electrolyte before and after exposure to an analytemolecule (e.g., NO.) can be used to determine if the molecule adsorbs onthe electrode. Measurable changes in capacitance are expected when theanalyte adsorbs strongly on the electrode.

When a potential pulse is applied to an electrode, a current responsewill arise due to charging or discharging of the double layer. At shorttimes (tens of milliseconds or less) the total current measured will becomprised exclusively of this charging current. Charging current (i_(c))is given by the expression:

i _(c)=(ΔE/R)×exp(−t/RC)

where ΔE is the magnitude of the potential pulse applied (in Volts), Ris the solution resistance (in ohms), t is time (in sec), and C is thedouble layer capacitance (in Farads). Thus, double layer capacitance isdetermined by plotting the logarithm (In) of charging current versustime according to:

In(i _(c))=In(ΔE/R)−(t/RC)

Since ΔE is known (experimentally specified in this case), they-intercept from the above procedure gives the solution resistance, andthe double layer capacitance can be obtained from the slope of the plot.The instrumentation used in these experiments (a BAS 100B potentiostat)actually implements the above experiment in the following way. Asymmetrical, square wave voltage pulse, centered about a user-selecteddc-offset potential (dc ±25 mV), is applied. Two current samples arethen taken; one at 54 microseconds (μs) after imposition of thepotential pulse and another at 72 μs after the pulse. The value of thesolution resistance is then obtained by extrapolation to t=0 of thebest-fit line through the two values for current. This procedure(imposition of a pulse, taking of two current measurements, andextrapolation to t=0) is automatically repeated 256 times, and a meanvalue for R is obtained. A mean time constant (RC) is also obtained,defined as the time needed for the current to decay to 37% of itsinitial, instantaneous value.

First experimental series. This set of experiments was conducted todetermine if the capacitance of an oxide-covered Ru electrode is alteredby exposure to NO. (The oxide film in these experiments was grown byholding the electrode at a potential of 675 mV for 2 hours, but couldpresumably be generated by chemical oxidation, instead.) In theseexperiments: (1) A 500 μm-diameter Ru electrode was polished, and itscapacitance (C) measured at a variety of dc-offset potentials. (2) Theelectrode potential was then held at ±675 mV vs. the Ag/AgCl referencefor 2 hours (the so-called “Quiet Time” or QT). The electrodecapacitance was then re-measured. (3) The experiment was repeated, thistime exposing the electrode after the QT to 10% NO. ( by maintaining 10%NO. in the headspace above the analyte solution), measuring thecapacitance again at each dc-offset potential, and calculating the meanvalue of C. (4) Finally, the experiment was conducted once again, thistime exposing the electrode to 10% NO. for 30 minutes after the QT andthen purging with N₂ for 30 minutes, and the mean value of C was thendetermined as before.

The table below summarizes these data and includes control data for a Ptelectrode. Specifically, the data below are initial and final values formean capacitance in microFarads (μF) of 500 μm diameter Ru and Ptelectrodes, with the values shown in parentheses being the percent (%)difference between initial and final capacitance measurements.

After After Quiet After After Polishing Time (QT) QT + NO· QT + NO· + N₂Ruthenium: 0.290 μF 0.589 μF (+103%) 0.326 μF 0.378 μF (+16%) 0.419 μF0.815 μF (+95%) Platnium: 0.252 μF 0.252 μF   (0%)

As is evident from these data, a measurable and significant increase incapacitance occurs in the Ru electrode after the QT, but not in the Ptelectrode. Also, the reversible decrease in capacitance of the Ruelectrode that occurs after exposure to NO. is evident.

Several conclusions may be drawn from the first experimental series: (1)Polarizing a Ru electrode at ±675 mV for 2 hours results in asignificant increase in the measured capacitance which could, inprinciple, be caused either by formation of an oxide film on the surfaceor by potentiostatic surface roughening, which could increase themicroscopic surface area of the electrode. The former explanation isfavored, as discussed below. (2) On the other hand, polarizing a Ptelectrode at +800 mV (the potential most likely to show an effect on Pt)for 2 hours does not appear to affect the measured electrodecapacitance. (3) Exposure to 10% NO. of an Ru electrode which haspreviously been held ±675 mV for 2 hours causes the measured electrodecapacitance to return to a value nearly equal to that for a freshlypolished electrode. This strongly suggests that the 2-hour quiet time(polarization at ±675 mV) causes formation of an oxide layer rather thana change in surface roughness (i.e. a change in microscopic area). Thatis, there is no reason to expect that exposure of roughened Ru to NO.would smooth the surface. (4) The fact that the capacitance of theoxide-covered Ru electrode decreases after exposure to NO. providesevidence of a specific chemical interaction between NO. and theelectrode surface. This interaction is apparently reversible, since theadsorbed NO. can be displaced by N₂. That is, nitrogen saturation of asolution into which the NO.-treated Ru electrode is immersed results inrestoration of the capacitance value that had previously been observedafter 2-hours of polarization (QT). (5) The results of this first seriesof experiments, when taken together, provide strong electrochemicalevidence for the existence of a surface complex (or complexes) formingbetween NO. in a saline medium and an Ru electrode, but not with a Ptelectrode. Apparently, this complexation only occurs if the surface ofthe Ru electrode has previously been partially oxidized.

Second experimental series. This set of experiments was conducted todetermine if the presence of NO. affects the oxide film grown on thesurface of the Ru electrode during the QT.

The formation and destruction of oxide films on electrode surfaces canalso be probed directly using the electrochemical technique calledcyclic voltammetry. In this technique, the voltage of the Ru electroderelative to a reference electrode (Ag/AgCl in this case) is caused tochange and the resulting current is monitored. Forcing the voltage to apositive value drives formation of the oxide layer, and an anodic(oxidation) current is registered. Conversely, when a negative voltageis applied, the oxide film is reduced to regenerate the “bare” Ru metal,and a cathodic (reduction) current is observed. As will be shown below,the interaction of NO. with the surface of an Ru electrode preventsreduction of the oxide layer. This is indicative of the formation of aninner coordination sphere complex between NO. and the Ru surface, thusshifting the reduction potential of the Ru—O functional groups on thesurface.

Voltammogram (A) of accompanying FIG. 3 was obtained at an Ru electrodeimmersed in nitrogen-saturated phosphate buffered saline (PBS). Theupward-going peaks appearing at about −175 and −245 mV are due to thereduction to elemental Ru of various oxides (Ru—O_(x), where x is apositive number greater than 0, for example, between 0 up to andincluding 2 and greater.). Voltammogram (B) of accompanying FIG. 3 wasobtained after saturation of the saline solution with 10% NO. and showsthat reduction of Ru is greatly inhibited in the presence of NO.

The voltammograms in FIG. 3 show clear interactions between NO. and theoxide layer on a Ru electrode. The upward-going peaks that appear atabout −175 and −245 mV are due to the reduction to elemental Ru ofvarious Ru oxides. As can clearly be seen, these upward-going(reduction) peaks in the voltammogram A obtained using an Ru electrodeimmersed in nitrogen-saturated PBS have largely disappeared involtammogram B obtained using the Ru electrode in PBS saturated with 10%NO., and the greatly-diminished remnants of those peaks have shifted tomore negative potentials. This indicates a chemical change in theRu—O_(x) layer due to the presence of NO. For example, formation of anadlayer of Ru—O—NO or Ru(Cl_(x))—O—NO having a more negative reductionpotential than the original Ru—O_(x) would be consistent with theseresults.

The conclusion reached from this second series of experiments, togetherwith the capacitance data from the first experimental series, is thatpartial oxidation of an Ru electrode in aqueous saline creates a surfaceonto which NO. can adsorb and be easily detected by electrochemicalmeans. Our previous data have shown that polarization of the electrodeat +675 mV is optimal for formation of the useful oxide layer. Use ofmore positive potentials results in a drastically diminishedsensitivity. Furthermore, published Pourbaix diagrams for Ru (that is,diagrams of potential vs. pH; see J. F. Llopis et al, “Ruthenium,”chapter VI-8, A. J. Bard, Encyclopedia of Electrochemistry of theElements, Marcel Dekker, Inc. (1976), the entire content of which isexpressly incorporated hereinto by reference) lead us to suspect thatthe active oxide is not RuO₂, but must consist of acontinuously-variable mixture of elemental ruthenium and its oxidesRuO_(x), where x is a positive number from just above 0 through higheroxidation states, even above 2, and includes fractional values.

EXAMPLE III

In order to demonstrate electrode efficacy in a complex biological fluidcontaining proteins, a ruthenium working electrode, an Ag/AgCl referenceelectrode, and a Pt auxiliary electrode were employed in anelectrochemical cell containing a 2 ml stirred sample of human bloodplasma to which 1.5 μM nitric oxide was added. The gas flowingcontinuously in the headspace above the stirred sample was 100% N₂ whichwas periodically alternated with 0.1% NO. in N₂. The original bloodsample was collected in a Vacutainer™ sample container (Becton Dickinson& Co.) in Li-heparin. As is evident from the plot of accompanying FIG.4, the electrode of this invention accurately detected the presence ofnitric oxide in the sample.

While the invention has been described in connection with what ispresently considered to be the most practical and preferred embodiment,it is to be understood that the invention is not to be limited to thedisclosed embodiment, but on the contrary, is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

What is claimed is:
 1. An electrode having a surface region comprised ofa metal oxide capable of enhanced detection of nitric oxide when exposedto a nitric oxide-containing fluid.
 2. The electrode of claim 1, whereinat least the surface region comprised of at least one oxide ofruthenium.
 3. The electrode of claim 1, wherein said at least onesurface region is comprised of at least one oxide of ruthenium havingthe formula RuO_(x), where x is a number greater than
 0. 4. Theelectrode of claim 3, wherein x is a number greater than 0 up to andincluding about
 2. 5. The electrode of claim 1, wherein at least thesurface region consists essentially of ruthenium conditioned at apotential for a time sufficient to cause the surface region to exhibitmaximal nitric oxide response when exposed to the nitricoxide-containing fluid.
 6. A nitric-oxide specific electrode having asurface region conditioned at a potential which enhances nitric oxidedetection when the electrode is exposed to a nitric oxide-containingfluid.
 7. The electrode of claim 6, wherein said at least one oxide ofruthenium is RuO_(x), where x is a number greater than
 0. 8. Theelectrode of claim 7, wherein where x is greater than 0 up to andincluding about
 2. 9. A medical device which comprises an electrodeaccording to any one of claims 1-8.
 10. The device of claim 9, adaptedto detect nitric oxide in a biological fluid.
 11. The device of claim10, wherein the biological fluid is at least one of blood, urine andsynovial fluid.
 12. A medical device as in claim 9 adapted for the invivo detection of nitric oxide in a biological fluid.
 13. A method forthe in vivo detection of nitric oxide comprising placing an electrodeaccording to any one of claims 1-8 at a site within a patient or otherliving organism, and then determining the nitric oxide present at saidsite by the electrochemical response of said electrode.
 14. A method ofdetecting nitric oxide in a biological sample comprising bringing anelectrode according to any one of claims 1-8 into contact with thebiological sample, and then determining the nitric oxide present in saidsample by the electrochemical response of said electrode.
 15. A methodof making an electrode for the detection of nitric oxide which comprisesconditioning a metal electrode surface at a potential for a timesufficient for the surface to exhibit enhanced nitric oxide response.16. The method as in claim 15, wherein the metal electrode surface isheld at a potential for a time sufficient to allow electrode current todecay to a stable baseline.
 17. An electrode for the detection of nitricoxide made according to claim 15 or 16.